We will extend our successful forward genetic analysis of susceptibility to mouse cytomegalovirus (MCMV) nfection, and institute a new screen for cell-autonomous resistance to Rift Valley Fever Virus (RVFV). These studies will build upon technological advances that permit us to find mutations faster than ever before, using massively parallel sequencing, both by itself and in combination with inducible pluripotent stem cell (IPS) technology in screens for cell-autonomous resistance to RVFV in mouse embryonic fibroblasts; MEFs. We have found that it is possible to screen mutagenized MEFs from reprogrammable C57BL/6J mice for resistance to RVFV ex vivo, to identify resistant clones, to use these clones to regenerate live mice, and at the same time, detect most of the mutations in these cells by sequencing genomic DNA and/or whole exomes. We have determined, based on in silico simulations, that compound heterozygous null alleles at almost all loci will result from mutagenesis on the scale we contemplate. As such, we hope to enrich our understanding of what it takes to combat a model herpesvirus infection with strong relevance to a human disease (HCMV infection), and also, to determine the critical host proteins necessary for a Category A pathogen (RVFV) to proliferate in mammalian cells. This work, pursued in depth, will inform us of molecular targets for the treatment of bunyavirus infections in general. In a third, circumscribed specific aim, we will analyze a new mutation, identified in screening, that abolishes the responses of plasmacytoid dendritic cells (PDCs) to nucleic acids. This mutation, called feeble, affects a channel protein that acts in conjunction with the adaptor protein 3 (APS) complex to condition endosomes in PDCs (but not other cells), making them competent to signal via TLRs. Based on our studies to date, we infer the existence of an ARF1->AP3^Slc45a->TLR7/9 pathway essential for the support of type I interferon gene induction in PDCs. The further elucidation of this pathway will deepen understanding of PDC function, and the role of PDC- derived interferon both in infection and in pathological settings such as systemic lupus erythematosus. RELEVANCE (See instructions): Project 1 will contribute to human health by finding key genes required for combating a model herpesvirus infection (mouse cytomegalovirus, similar to human cytomegalovirus, a major pathogen). It will also contribute by finding genes needed by Rift Valley Fever Virus (RVFV) to complete its life cycle in the mammalian host. Human cytomegalovirus is a major cause of blindness, neurological disease, and birth defects in humans, while RVFV is a major economic problem in Africa with some crossover of disease into human populations.